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1.
Phys Chem Chem Phys ; 22(11): 6136-6144, 2020 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-32124883

RESUMO

Histone lysine methylation regulates the recruitment of mammalian DNA repair factor 53BP1 to the histone H4 lysine 20 (H4K20), through specific recognition of the tandem Tudor domain of 53BP1. The di- and mono-methylated H4K20 bind to 53BP1 with high affinity, but the non- and tri-methylated H4K20 do not. Here, we develop a new approach to carry out computational study to unravel the binding mechanism of methylated H4K20 by 53BP1 and to compute relative binding affinities of different methylations of H4K20 by 53BP1. First, hot spots in 53BP1 were predicted by computational alanine scanning and aromatic cages formed by W1495, Y1500, Y1502, and Y1523 are found to provide the dominant binding to di- and mono-methylated H4K20 in addition to D1521. Secondly, a de-methylation method is proposed to predict relative binding free energies between 53BP1 and different methylated states of H4K20. Finally, the tri-methylated and non-methylated H4K20/53BP1 complexes are found to be dynamically unstable, explaining the experimental finding that neither can bind to 53BP1. The present work provides an important theoretical basis for our understanding of histone methylations of H4K20 and their recognition mechanism by DNA repair factor 53BP1.

2.
J Chem Inf Model ; 60(3): 1245-1252, 2020 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-32126171

RESUMO

Computational protein design remains a challenging task despite its remarkable success in the past few decades. With the rapid progress of deep-learning techniques and the accumulation of three-dimensional protein structures, the use of deep neural networks to learn the relationship between protein sequences and structures and then automatically design a protein sequence for a given protein backbone structure is becoming increasingly feasible. In this study, we developed a deep neural network named DenseCPD that considers the three-dimensional density distribution of protein backbone atoms and predicts the probability of 20 natural amino acids for each residue in a protein. The accuracy of DenseCPD was 53.24 ± 0.17% in a 5-fold cross-validation on the training set and 55.53% and 50.71% on two independent test sets, which is more than 10% higher than those of previous state-of-the-art methods. Two approaches for using DenseCPD predictions in computational protein design were analyzed. The approach using the cutoff of accumulative probability had a smaller sequence search space compared with the approach that simply uses the top-k predictions and therefore enabled higher sequence identity in redesigning three proteins with Rosetta. The network and the datasets are available on a web server at http://protein.org.cn/densecpd.html. The results of this study may benefit the further development of computational protein design methods.

3.
Nucleic Acids Res ; 48(D1): D320-D327, 2020 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-31906602

RESUMO

Liquid-liquid phase separation (LLPS) leads to a conversion of homogeneous solution into a dense phase that often resembles liquid droplets, and a dilute phase. An increasing number of investigations have shown that biomolecular condensates formed by LLPS play important roles in both physiology and pathology. It has been suggested the phase behavior of proteins would be not only determined by sequences, but controlled by micro-environmental conditions. Here, we introduce LLPSDB (http://bio-comp.ucas.ac.cn/llpsdb or http://bio-comp.org.cn/llpsdb), a web-accessible database providing comprehensive, carefully curated collection of proteins involved in LLPS as well as corresponding experimental conditions in vitro from published literatures. The current release of LLPSDB incorporates 1182 entries with 273 independent proteins and 2394 specific conditions. The database provides a variety of data including biomolecular information (protein sequence, protein modification, nucleic acid, etc.), specific phase separation information (experimental conditions, phase behavior description, etc.) and comprehensive annotations. To our knowledge, LLPSDB is the first available database designed for LLPS related proteins specifically. It offers plenty of valuable resources for exploring the relationship between protein sequence and phase behavior, and will enhance the development of phase separation prediction methods, which may further provide more insights into a comprehensive understanding of LLPS in cellular function and related diseases.

4.
J Comput Chem ; 41(5): 415-420, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31329318

RESUMO

The double electron-electron resonance (DEER) is a powerful structural biology technique to obtain distance information in the range of 18 to 80 å by measuring the dipolar coupling between two unpaired electron spins. The distance distributions obtained from the experiment provide valuable structural information about the protein in its native environment that can be exploited using restrained ensemble molecular dynamics (reMD) simulations. We present a new tool DEER Facilitator in CHARMM-GUI that consists of two modules Spin-Pair Distributor and reMD Prepper to setup simulations that utilize information from DEER experiments. Spin-Pair Distributor provides a web-based interface to calculate the spin-pair distance distribution of labeled sites in a protein using MD simulations. The calculated distribution can be used to guide the selection of the labeling sites in experiments as well as validate different protein structure models. reMD Prepper facilities the setup of reMD simulations using different types of spin labels in four different environments including vacuum, solution, micelle, and bilayer. The applications of these two modules are demonstrated with several test cases. Spin-Pair Distributor and reMD Prepper are available at http://www.charmm-gui.org/input/deer and http://www.charmm-gui.org/input/deerre. DEER Facilitator is expected to facilitate advanced biomolecular modeling and simulation, thereby leading to an improved understanding of the structure and dynamics of complex biomolecular systems based on experimental DEER data. © 2019 Wiley Periodicals, Inc.

5.
Chemphyschem ; 21(3): 263-271, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31816138

RESUMO

Polycomb Polycomb repressive complex 2 (PRC2) plays a key role in silencing epigenetic gene through trimethylation of lysine 27 on histone 3 (H3K27). Dysregulations of PRC2 caused by overexpression and mutations of the core subunits of PRC2 have been implicated in many cancers. The core subunits EZH1/2 are histone-lysine N-methyltransferases that function as the enzymatic component of PRC2. While the core subunit EED is a scaffolding protein to support EZH1/2 and binds JARID2K116me3/H3K27me3 to enhance the enzymatic activity of PRC2 through allosteric activation. Recently, several small molecules that compete with JARI2K116me3 and H3K27me3 have been reported. These molecules selectively bind to the JARID2K116me3/H3K27me3-binding pocket of EED, thereby preventing the allosteric regulation of PRC2. These first-in-class PRC2 inhibitors show robust suppression in DLBCL cell lines, demonstrating anticancer drugs that target the EED subunit of PRC2 are viable. In this study, we used the recently developed MM/GBSA_IE and the alanine scanning method to analyze the hot spots in EED/inhibitor interactions. The analysis of these hot and warm spots helps us to understand the fundamental differences between inhibitors. Our results give a quantitative explanation on why the binding affinities of EED/A-395 interactions are stronger than that of EED/EED226 while their binding modes are similar and provide valuable insights for rational design of novel EED inhibitors.


Assuntos
Indanos/metabolismo , Complexo Repressor Polycomb 2/antagonistas & inibidores , Complexo Repressor Polycomb 2/metabolismo , Sulfonamidas/metabolismo , Sulfonas/metabolismo , Triazóis/metabolismo , Sítios de Ligação , Humanos , Ligantes , Simulação de Acoplamento Molecular , Complexo Repressor Polycomb 2/química , Ligação Proteica , Termodinâmica
6.
J Chem Inf Model ; 59(9): 3871-3878, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31442042

RESUMO

Mitochondrial serine hydroxymethyl transferase isoform 2 (SHMT2) has attracted increasing attention as a pivotal catalyzing regulator of the serine/glycine pathway in the one-carbon metabolism of cancer cells. However, few inhibitors that target this potential anticancer target have been discovered. Quantitative characterization of the interactions between SHMT2 and its known inhibitors should benefit future discovery of novel inhibitors. In this study, we employed a recently developed alanine-scanning-interaction-entropy method to quantitatively calculate the residue-specific binding free energy of 28 different SHMT2 inhibitors that originate from the same skeleton. Major contributing residues from SHMT2 and chemical groups from the inhibitors were identified, and the binding energy of each residue was quantitatively determined, revealing essential features of the protein-inhibitor interaction. The most important contributing residue is Y105 of the B chain followed by L166 of the A chain. The calculated protein-ligand binding free energies are in good agreement with the experimental results and showed better correlation and smaller errors compared with those obtained using the conventional MM/GBSA with the normal mode method. These results may aid the rational design of more effective SHMT2 inhibitors.

7.
J Chem Inf Model ; 59(4): 1508-1514, 2019 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-30759982

RESUMO

Accurately predicting changes in protein stability due to mutations is important for protein engineering and for understanding the functional consequences of missense mutations in proteins. We have developed DeepDDG, a neural network-based method, for use in the prediction of changes in the stability of proteins due to point mutations. The neural network was trained on more than 5700 manually curated experimental data points and was able to obtain a Pearson correlation coefficient of 0.48-0.56 for three independent test sets, which outperformed 11 other methods. Detailed analysis of the input features shows that the solvent accessible surface area of the mutated residue is the most important feature, which suggests that the buried hydrophobic area is the major determinant of protein stability. We expect this method to be useful for large-scale design and engineering of protein stability. The neural network is freely available to academic users at http://protein.org.cn/ddg.html .

8.
J Comput Chem ; 40(7): 893-899, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30677169

RESUMO

Nanodiscs are discoidal protein-lipid complexes that have wide applications in membrane protein studies. Modeling and simulation of nanodiscs are challenging due to the absence of structures of many membrane scaffold proteins (MSPs) that wrap around the membrane bilayer. We have developed CHARMM-GUI Nanodisc Builder (http://www.charmm-gui.org/input/nanodisc) to facilitate the setup of nanodisc simulation systems by modeling the MSPs with defined size and known structural features. A total of 11 different nanodiscs with a diameter from 80 to 180 Å are made available in both the all-atom CHARMM and two coarse-grained (PACE and Martini) force fields. The usage of the Nanodisc Builder is demonstrated with various simulation systems. The structures and dynamics of proteins and lipids in these systems were analyzed, showing similar behaviors to those from previous all-atom and coarse-grained nanodisc simulations. We expect the Nanodisc Builder to be a convenient and reliable tool for modeling and simulation of nanodisc systems. © 2019 Wiley Periodicals, Inc.

9.
Glycobiology ; 29(4): 320-331, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30689864

RESUMO

Characterizing glycans and glycoconjugates in the context of three-dimensional structures is important in understanding their biological roles and developing efficient therapeutic agents. Computational modeling and molecular simulation have become an essential tool complementary to experimental methods. Here, we present a computational tool, Glycan Modeler for in silico N-/O-glycosylation of the target protein and generation of carbohydrate-only systems. In our previous study, we developed Glycan Reader, a web-based tool for detecting carbohydrate molecules from a PDB structure and generation of simulation system and input files. As integrated into Glycan Reader in CHARMM-GUI, Glycan Modeler (Glycan Reader & Modeler) enables to generate the structures of glycans and glycoconjugates for given glycan sequences and glycosylation sites using PDB glycan template structures from Glycan Fragment Database (http://glycanstructure.org/fragment-db). Our benchmark tests demonstrate the universal applicability of Glycan Reader & Modeler to various glycan sequences and target proteins. We also investigated the structural properties of modeled glycan structures by running 2-µs molecular dynamics simulations of HIV envelope protein. The simulations show that the modeled glycan structures built by Glycan Reader & Modeler have the similar structural features compared to the ones solved by X-ray crystallography. We also describe the representative examples of glycoconjugate modeling with video demos to illustrate the practical applications of Glycan Reader & Modeler. Glycan Reader & Modeler is freely available at http://charmm-gui.org/input/glycan.


Assuntos
Carboidratos/química , Biologia Computacional , Glicoconjugados/química , Polissacarídeos/química , Configuração de Carboidratos , Bases de Dados Factuais
10.
J Chem Theory Comput ; 15(1): 775-786, 2019 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-30525595

RESUMO

Glycolipids (such as glycoglycerolipids, glycosphingolipids, and glycosylphosphatidylinositol) and lipoglycans (such as lipopolysaccharides (LPS), lipooligosaccharides (LOS), mycobacterial lipoarabinomannan, and mycoplasma lipoglycans) are typically found on the surface of cell membranes and play crucial roles in various cellular functions. Characterizing their structure and dynamics at the molecular level is essential to understand their biological roles, but systematic generation of glycolipid and lipoglycan structures is challenging because of great variations in lipid structures and glycan sequences (i.e., carbohydrate types and their linkages). To facilitate the generation of all-atom glycolipid/LPS/LOS structures, we have developed Glycolipid Modeler and LPS Modeler in CHARMM-GUI ( http://www.charmm-gui.org ), a web-based interface that simplifies building of complex biological simulation systems. In addition, we have incorporated these modules into Membrane Builder so that users can readily build a complex symmetric or asymmetric biological membrane system with various glycolipids and LPS/LOS. These tools are expected to be useful in innovative and novel glycolipid/LPS/LOS modeling and simulation research by easing tedious and intricate steps in modeling complex biological systems and shall provide insight into structures, dynamics, and underlying mechanisms of complex glycolipid-/LPS-/LOS-containing biological membrane systems.


Assuntos
Glicolipídeos/química , Lipopolissacarídeos/química , Proteínas de Bactérias/química , Antígenos CD59/química , Campylobacter jejuni/química , Membrana Celular/química , Simulação por Computador , Escherichia coli/química , Glicosilfosfatidilinositóis/química , Humanos , Simulação de Dinâmica Molecular , Interface Usuário-Computador
11.
J Comput Chem ; 40(9): 1045-1056, 2019 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-30549062

RESUMO

The recently developed MM/GBSA_IE method is applied to computing hot and warm spots in p53/PMI-MDM2/MDMX protein-protein interaction systems. Comparison of the calculated hot (>2 kcal/mol) and warm spots (>1 kcal/mol) in P53 and PMI proteins interacting with MDM2 and MDMX shows a good quantitative agreement with the available experimental data. Further, our calculation predicted hot spots in MDM2 and MDMX proteins in their interactions with P53 and PMI and they help elucidate the interaction mechanism underlying this important PPI system. In agreement with the experimental result, the present calculation shows that PMI has more hot and warm spots and binds stronger to MDM2/MDMX. The analysis of these hot and warm spots helps elucidate the fundamental difference in binding between P53 and PMI to the MDM2/MDMX systems. Specifically, for p53/PMI-MDM2 systems, p53 and PMI use essentially the same residues (L54, I61, Y67, Q72, V93, H96, and I99) of MDM2 for binding. However, PMI enhanced interactions with residues L54, Y67, and Q72 of MDM2. For the p53/PMI-MDMX system, p53 and PMI use similar residues (M53, I60, Y66, Q71, V92, and Y99) of MDMX for binding. However, PMI exploited three extra residues (M61, K93, and L98) of MDMX for enhanced binding. In addition, PMI enhanced interaction with four residues (M53, Y66, Q71, and Y99) of MDMX. These results gave quantitative explanation on why the binding affinities of PMI-MDM2/MDMX interactions are stronger than that of p53-MDM2/MDMX although their binding modes are similar. © 2018 Wiley Periodicals, Inc.

12.
Breast Cancer Res ; 20(1): 116, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30285809

RESUMO

BACKGROUND: Increased collagen expression and deposition are associated with cancer progression and poor prognosis in breast cancer patients. However, function and regulation of membrane-associated collagen in breast cancer have not been determined. Collagen XIII is a type II transmembrane protein within the collagen superfamily. Experiments in tissue culture and knockout mouse models show that collagen XIII is involved in cell adhesion and differentiation of certain cell types. In the present study, we determined roles of collagen XIII in breast cancer progression and metastasis. METHODS: We analyzed the association of collagen XIII expression with breast cancer development and metastasis using published gene expression profiles generated from human breast cancer tissues. Utilizing gain- and loss- of function approaches and 3D culture assays, we investigated roles of collagen XIII in regulating invasive tumor growth. Using the tumorsphere/mammosphere formation assay and the detachment cell culture assay, we determined whether collagen XIII enhances cancer cell stemness and induces anoikis resistance. We also inhibited collagen XIII signaling with ß1 integrin function-blocking antibody. Finally, using the lung colonization assay and the orthotopic mammary tumor model, we investigated roles of collagen XIII in regulating breast cancer colonization and metastasis. Cox proportional hazard (log-rank) test, two-sided Student's t-test (two groups) and one-way ANOVA (three or more groups) analyses were used in this study. RESULTS: Collagen XIII expression is significantly higher in human breast cancer tissue compared with normal mammary gland. Increased collagen XIII mRNA levels in breast cancer tissue correlated with short distant recurrence free survival. We showed that collagen XIII expression promoted invasive tumor growth in 3D culture, enhanced cancer cell stemness, and induced anoikis resistance. Collagen XIII expression induced ß1 integrin activation. Blocking ß1 integrin activation significantly reduced collagen XIII-induced invasion and mammosphere formation. Importantly, silencing collagen XIII in MDA-MB-231 cells reduced lung colonization and metastasis. CONCLUSIONS: Our results demonstrate a novel function of collagen XIII in promoting cancer metastasis, cell invasion, and anoikis resistance.


Assuntos
Anoikis , Neoplasias da Mama/metabolismo , Colágeno Tipo VIII/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/terapia , Linhagem Celular , Linhagem Celular Tumoral , Colágeno Tipo VIII/genética , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Proteínas de Membrana/genética , Camundongos SCID , Interferência de RNA , Terapêutica com RNAi/métodos , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
13.
Front Cell Dev Biol ; 6: 66, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30003082

RESUMO

Collagen is the major component of extracellular matrix. Collagen cross-link and deposition depend on lysyl hydroxylation, which is catalyzed by procollagen-lysine, 2-oxoglutarate 5-dioxygenase (PLOD). Aberrant lysyl hydroxylation and collagen cross-link contributes to the progression of many collagen-related diseases, such as fibrosis and cancer. Three lysyl hydroxylases (LH1, LH2, and LH3) are identified, encoded by PLOD1, PLOD2, and PLOD3 genes. Expression of PLODs is regulated by multiple cytokines, transcription factors and microRNAs. Dysregulation of PLODs promotes cancer progression and metastasis, suggesting that targeting PLODs is potential strategy for cancer treatment. Here, we summarize the recent progress in the investigation of function and regulation of PLODs in normal tissue development and disease progression, especially in cancer.

14.
Sci Rep ; 8(1): 6349, 2018 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-29679026

RESUMO

Computational protein design has a wide variety of applications. Despite its remarkable success, designing a protein for a given structure and function is still a challenging task. On the other hand, the number of solved protein structures is rapidly increasing while the number of unique protein folds has reached a steady number, suggesting more structural information is being accumulated on each fold. Deep learning neural network is a powerful method to learn such big data set and has shown superior performance in many machine learning fields. In this study, we applied the deep learning neural network approach to computational protein design for predicting the probability of 20 natural amino acids on each residue in a protein. A large set of protein structures was collected and a multi-layer neural network was constructed. A number of structural properties were extracted as input features and the best network achieved an accuracy of 38.3%. Using the network output as residue type restraints improves the average sequence identity in designing three natural proteins using Rosetta. Moreover, the predictions from our network show ~3% higher sequence identity than a previous method. Results from this study may benefit further development of computational protein design methods.


Assuntos
Engenharia de Proteínas/métodos , Algoritmos , Sequência de Aminoácidos , Aminoácidos , Big Data , Biologia Computacional , Aprendizado Profundo , Aprendizado de Máquina , Probabilidade , Proteínas/metabolismo
15.
Nat Chem Biol ; 14(5): 489-496, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29581582

RESUMO

ClC chloride channels and transporters are important for chloride homeostasis in species from bacteria to human. Mutations in ClC proteins cause genetically inherited diseases, some of which are likely to involve folding defects. The ClC proteins present a challenging and unusual biological folding problem because they are large membrane proteins possessing a complex architecture, with many reentrant helices that go only partway through membrane and loop back out. Here we were able to examine the unfolding of the Escherichia coli ClC transporter, ClC-ec1, using single-molecule forced unfolding methods. We found that the protein could be separated into two stable halves that unfolded independently. The independence of the two domains is consistent with an evolutionary model in which the two halves arose from independently folding subunits that later fused together. Maintaining smaller folding domains of lesser complexity within large membrane proteins may be an advantageous strategy to avoid misfolding traps.


Assuntos
Canais de Cloreto/química , Cloretos/química , Escherichia coli/química , DNA/química , Dimiristoilfosfatidilcolina/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Evolução Molecular , Humanos , Proteínas de Membrana Transportadoras/química , Simulação de Dinâmica Molecular , Mutação , Plasmídeos , Desnaturação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Estrutura Secundária de Proteína
16.
Oncotarget ; 9(18): 14124-14137, 2018 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-29581832

RESUMO

The underlying cause of treatment failure in many cancer patients is intrinsic and acquired resistance to chemotherapy. Recently, histone deacetylase (HDAC) inhibitors have developed into a promising cancer treatment. However, resistance mechanism induced by HDAC inhibitors remains largely unknown. Here we report that a HDAC inhibitor, JNJ-2648158 induced transcription of XIAP by activating AP-1 expression, which conferring resistance to chemotherapeutics. Our results showed that high expression of c-Fos caused by HDAC inhibitor promoted AP-1 formation during acquired resistance towards chemo-drugs, indicating an extremely poor clinical outcome in breast cancers and liver cancers. Our study reveals a novel regulatory mechanism towards chemo-drug resistance, and suggests that XIAP may serve as a potential therapeutic target in those chemo-resistant cancer cells.

17.
Cancer Res Treat ; 50(3): 894-907, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28934847

RESUMO

Purpose: Cancer-associated fibroblasts (CAFs) activated by cancer cells has a central role in development and malignant biological behavior in colorectal cancer (CRC). Adult fibroblasts do not express Snail, but Snail-positive fibroblasts are discovered in the stroma of malignant CRC and reported to be the key role to chemoresistance. However, the reciprocal effect of CAFs expressed Snail to chemoresistance on CRC cells and the underlying molecular mechanisms are not fully characterized. Materials and Methods: Snail-overexpressed 3T3 stable cell lines were generated by lipidosome and CT26 mixed with 3T3-Snail subcutaneous transplanted CRC models were established by subcutaneous injection. Cell Counting Kit-8, flow cytometry and western blotting assays were performed, and immunohistochemistry staining was studied. The cytokines participated in chemoresistance was validated with reverse transcriptase-polymerase chain reaction and heatmap. Results: Snail-expression fibroblasts are discovered in human and mouse spontaneous CRCs. Overexpression of Snail induces 3T3 fibroblasts transdifferentiation to CAFs. CT26 co-cultured with 3T3-Snail resisted the impairment from 5-fluorouracil and paclitaxel in vitro. The subcutaneous transplanted tumor models included 3T3-Snail cells develop without restrictions even after treating with 5-fluorouracil or paclitaxel. Moreover, these chemoresistant processes may be mediated by CCL1 secreted by Snail-expression fibroblasts via transforming growth factor ß/nuclear factor-κB signaling pathways. Conclusion: Taken together, Snail-expressing 3T3 fibroblasts display CAFs properties that support 5-fluorouracil and paclitaxel chemoresistance in CRC via participation of CCL1 and suggest that inhibition of the Snail-expression fibroblasts in tumor may be a useful strategy to limit chemoresistance.


Assuntos
Fibroblastos Associados a Câncer/citologia , Quimiocina CCL1/metabolismo , Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fatores de Transcrição da Família Snail/metabolismo , Células 3T3 , Animais , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/metabolismo , Linhagem Celular Tumoral , Quimiocina CCL1/genética , Técnicas de Cocultura , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Fluoruracila/farmacologia , Humanos , Camundongos , NF-kappa B/metabolismo , Transplante de Neoplasias , Paclitaxel/farmacologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo
18.
J Phys Chem B ; 122(3): 1169-1175, 2018 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-29268602

RESUMO

The inherent flexibility of carbohydrates is dependent on stereochemical arrangements, and characterization of their influence and importance will give insight into the three-dimensional structure and dynamics. In this study, a ß-(1→4)/ß-(1→3)-linked glucosyl decasaccharide is experimentally investigated by synchrotron small-angle X-ray scattering from which its radius of gyration (Rg) is obtained. Molecular dynamics (MD) simulations of the decasaccharide show four populated states at each glycosidic linkage, namely, syn- and anti-conformations. The calculated Rg values from the MD simulation reveal that in addition to syn-conformers the presence of anti-ψ conformational states is required to reproduce experimental scattering data, unveiling inherent glycosidic linkage flexibility. The CHARMM36 force field for carbohydrates thus describes the conformational flexibility of the decasaccharide very well and captures the conceptual importance that anti-conformers are to be anticipated at glycosidic linkages of carbohydrates.


Assuntos
Glucanos/química , Simulação de Dinâmica Molecular , Configuração de Carboidratos , Espalhamento a Baixo Ângulo , Difração de Raios X
19.
Toxins (Basel) ; 9(10)2017 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-28937604

RESUMO

The anthrax lethal toxin consists of protective antigen (PA) and lethal factor (LF). Understanding both the PA pore formation and LF translocation through the PA pore is crucial to mitigating and perhaps preventing anthrax disease. To better understand the interactions of the LF-PA engagement complex, the structure of the LFN-bound PA pore solubilized by a lipid nanodisc was examined using cryo-EM. CryoSPARC was used to rapidly sort particle populations of a heterogeneous sample preparation without imposing symmetry, resulting in a refined 17 Å PA pore structure with 3 LFN bound. At pH 7.5, the contributions from the three unstructured LFN lysine-rich tail regions do not occlude the Phe clamp opening. The open Phe clamp suggests that, in this translocation-compromised pH environment, the lysine-rich tails remain flexible and do not interact with the pore lumen region.


Assuntos
Antígenos de Bactérias/ultraestrutura , Antraz , Toxinas Bacterianas , Microscopia Crioeletrônica , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína
20.
J Comput Chem ; 38(27): 2354-2363, 2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28776689

RESUMO

A complex cell envelope, composed of a mixture of lipid types including lipopolysaccharides, protects bacteria from the external environment. Clearly, the proteins embedded within the various components of the cell envelope have an intricate relationship with their local environment. Therefore, to obtain meaningful results, molecular simulations need to mimic as far as possible this chemically heterogeneous system. However, setting up such systems for computational studies is far from trivial, and consequently the vast majority of simulations of outer membrane proteins still rely on oversimplified phospholipid membrane models. This work presents an update of CHARMM-GUI Martini Maker for coarse-grained modeling and simulation of complex bacterial membranes with lipopolysaccharides. The qualities of the outer membrane systems generated by Martini Maker are validated by simulating them in bilayer, vesicle, nanodisc, and micelle environments (with and without outer membrane proteins) using the Martini force field. We expect this new feature in Martini Maker to be a useful tool for modeling large, complicated bacterial outer membrane systems in a user-friendly manner. © 2017 Wiley Periodicals, Inc.


Assuntos
Bactérias/química , Membrana Celular/química , Lipopolissacarídeos/química , Modelos Químicos , Desenho de Programas de Computador , Proteínas da Membrana Bacteriana Externa/química , Bicamadas Lipídicas/química , Micelas , Simulação de Dinâmica Molecular , Fosfolipídeos/química
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